We have previously investigated how the response regulator (RR) VpsR, together with the small molecule cyclic di-GMP (c-di-GMP) regulates V. chlolerae biofilm formation by characterizing VpsR-activated transcription from PvpsL, a promoter of biofilm biosynthesis genes. We found that while VpsR shares homology with enhancer binding proteins that activate sigma54-RNA polymerase, its activation of PvspL uses sigma70-RNA polymerase. Interestingly, c-di-GMP does not significantly change the affinity of VpsR for PvpsL DNA or the DNase I footprint of VpsR on the DNA, and it is not required for VpsR to dimerize. However, DNase I and KMnO4 footprints reveal that the sigma70-RNA polymerase / VpsR/c-di-GMP complex on PvpsL adopts a different conformation from that formed by sigma70-RNA polymerase alone, with c-di-GMP, or with VpsR. Our results suggested that c-di-GMP is required for VpsR to generate the specific protein-DNA architecture needed for activated transcription, a previously unrecognized role for c-di-GMP in gene expression. As VpsR is a RR, we have now investigated the importance of VpsR phosphorylation for PvpsL activity. Two-component signal transduction systems (TCSs), comprised of a sensor kinase (SK) that is phosphorylated and then transfers its phosphate to a RR, are used by bacteria to respond to environmental stimuli and extracellular signals. With 43 predicted SKs and 52 putative RRs, Vibrio cholerae is well-versed in environmental changes as it transitions between the aquatic reservoir and the human host. The highly conserved D59 residue of VpsR is predicted to be the potential site of phosphorylation. However, VpsR is an ophan RR , with no cognate SK upstream of the vpsR gene. To test the need for phosphorylation at D59, we compared the activities of the typically phosphomimic variant VpsR D59->E and the phosphodefective variant D59->A both in vivo and in vitro. We find that in the presence of c-di-GMP, either wildtype (WT) or the phosphomimic VpsR D59E activates PvpsL transcription, while the phosphodefective D59A variant does not. Although there is no known SK for VpsR and activation by purified WT VpsR in vitro does not require the addition of acetyl phosphate for activity, we demonstrate that a VpsR that is purified after denaturation/renaturation (VpsRren) is dependent on acetyl phosphate for its transcriptional activity, consistent with the idea that phosphorylation of D59 regulates VpsRs ability to activate transcription. Using gel retardation assays, BS3 crosslinking, trypsin digestion, and DNase I and KMnO4 footprints, we show that the presence of acetyl phosphate is needed for transcriptional activity in the presence of low levels of c-di-GMP, but is unnecessary in the presence of high levels of c-di-GMP. Our data suggest that c-di-GMP is required to for VpsR activation of transcription while phosphorylation of D59 modulates activity when c-di-GMP concentrations are low.